ES2240106T3 - USE OF BISPHOSPHONIC ACIDS FOR THE TREATMENT OF ANGIOGENESIS. - Google Patents

Abstract

Use of a bisphosphonate in the preparation of a medicament for the treatment of myocardial ischemia, osteoarthritis, psoriasis, hemangioblastoma, hemangioma or pain, where bisphosphonate acts as an agent of inhibition or reversal of angiogenesis and is selected from the following compounds or a pharmaceutically acceptable salt thereof, or any hydrate thereof: 3-amino-1- hydroxypropane-1,1-diphosphonic acid (pamidronic acid), eg pamidronate (APD); 3- (N, N-dimethylamino) -1-hydroxypropane-1,1-diphosphonic acid, e.g. dimethyl-APD; 4-amino-1- hydroxybutane-1,1-diphosphonic acid (alendronic acid), e.g. alendronate; 1-hydroxy-etidene bisphosphonic acid, e.g. etidronate; 1-hydroxy-3- (methylpentylamino) -propylidene bisphosphonic acid, ibandronic acid, e.g. ibandronate; 6-amino-1- hydroxyhexane-1, 1- diphosphonic acid, e.g. amino-hexyl-BP; 3- (N-methyl-N-n-pentylamino) -1- hydroxypropane-1,1-diphosphonic acid, e.g. methyl-pentyl-APD (= BM 21.0955); 1-hydroxy-2- (imidazol-1-yl-ethane-1, 1-diphosphonic acid, eg zoledronic acid; 1-hydroxy-2- (3- pyridyl) ethane -1,1-diphosphonic acid (risedronic acid), eg risedronate, including N-methyl pyridinium salts thereof, for example, N-methyl pyridinium iodides such as NE-10244 or NE-10446; 1- (4- chlorophenylthio acid ) methane-1,1-diphosphonic acid (tiludronic acid), eg tiludronate; 3- [N- (2- phenylthioethyl) -N-methylamino] -1- hydroxypropane-1, 1- diphosphonic acid; 1-hydroxy-3- acid (pyrrolidin-1-yl) propane-1,1-diphosphonic acid, eg EB 1053 (Leo); 1- (N-phenylaminothiocarbonyl) methane-1,1-diphosphonic acid, eg FR 78844 (Fujisawa); 5-benzoyl acid; -3, 4-dihydro-2H- pyrazol-3, 3-diphosphonic tetraethyl ester, eg U-81581 (Upjohn); 1-hydroxy-2- (imidazo [1,2-a] pyridin-3- yl) ethane -1, 1- diphosphonic, eg YM 529; and 1,1, 1- dichloromethane-1,1-diphosphonic acid (clodronic acid), eg clodronate.

Description

Use of bisphosphonic acids for treatment of angiogenesis.

This invention relates to new uses therapeutic bisphosphonates.

Bisphosphonates are widely used to inhibit osteoclast activity in a wide variety of both benign and malignant disorders in which bone resorption is increased. Yes, bisphosphonates have recently been available for long-term treatment of patients with Multiple Myeloma (MM). These pyrophosphate analogs not only reduce the occurrence of skeletal-related events but also provide patients with clinical benefits and improve survival. Bisphosphonates are able to prevent bone resorption in vivo ; The therapeutic efficacy of bisphosphonates has been demonstrated in the treatment of Pager's bone disease, tumor-induced hypercalcemia and, more recently, bone metastasis and multiple myeloma (MM) (for review, see Fleisch H 1997 Bisphosphonates clinical. In Bisphosphonates in Bone Disease. From the Laboratory to the Patient. Eds: The Parthenon Publishing Group, New York / London pp 68-163). The mechanisms by which bisphosphonates inhibit bone resorption are still poorly understood and appear to vary according to the bisphosphonates studied. It has been shown that bisphosphonates bind strongly to bone hydroxyapatite crystals, to reduce bone wear and resorption, to decrease levels of hydroxyproline or alkaline phosphatase in the blood and also to inhibit both the activation and activity of osteoclasts .

MM is a disease of the cells plasma characterized by the proliferation and accumulation of malignant plasma cells in the bone marrow. The main Clinical consequences are lytic bone lesions associated with pathological fractures and bone pain. Injuries are produced by excessive bone resorption, which frequently It leads to hypercalcemia. Bisphosphonates have been introduced for long-term treatment of MM in combination with the conventional chemotherapy It has been recently shown that bisphosphonates such as clodronate and pamidronate may reduce the occurrence of skeletal related events such as Lytic bone lesions and pathological fractures, and may relieve the bone pain and improve the quality of life of patients

It has been found now, surprisingly, that certain bisphosphonates have an embolic effect on the vessels newly formed capillary blood, which are formed during angiogenesis associated with tumor growth and invasion and some other pathological conditions such as inflammation, rheumatoid arthritis and osteoarthritis. In addition, it has been found that certain bisphosphonates angiogenesis induced by the factor of growth and endothelial cell proliferation in experiments with animal models and tissue culture.

In accordance with the foregoing, the present invention provides the use of a bisphosphonate in the preparation of a medication for the treatment of myocardial ischemia, osteoarthritis, psoriasis, hemangioblastoma, hemangioma or pain, in where bisphosphonate acts as an inhibitor or reversing agent of angiogenesis and is selected from the following compounds or of pharmaceutically acceptable salts thereof, or a hydrate thereof: acid 3-amino-1-hydroxypropane-1,1-diphosphonic (pamidronic acid), e.g. pamidronate (APD); acid 3- (N, N-dimethylamino) -1-hydroxypropane-1,1-diphosphonic,  e.g. dimethyl-APD; acid 4-amino-1-hydroxybutane-1,1-diphosphonic (alendronic acid), e.g. alendronate; acid 1-hydroxyeidene bisphosphonic, e.g. etidronate; acid 1-hydroxy-3- (methylpentylamino) -propylidene bisphosphonic, ibandronic acid, e.g. ibandronate; 6-amino-1-hydroxyhexane-1,1-diphosphonic acid e.g. amino-hexyl-BP; acid 3- (N-methyl-Nn-pentylamino) -1-hydroxypropane-1,1-diphosphonic, e.g. methyl-pentyl-APD (= BM 21.0955); 1-hydroxy-2- (imidazol-1-yl) ethane-1,1-diphosphonic acid e.g. Zoledronic acid; acid 1-hydroxy-2- (3-pyridyl) ethane-1,1-diphosphonic (risedronic acid), e.g. risedronate, including salts N-methyl pyridinium thereof, for example, N-methyl pyridinium iodides such as NE-10244 or NE-10446; acid 1- (4-chlorophenylthio) methane-1,1-diphosphonic (tiludronic acid), e.g. tiludronate; acid 3- [N- (2-phenylthioethyl) -N-methylamino] -1-hydroxypropane-1,1-diphosphonic; acid 1-hydroxy-3- (pyrrolidin-1-yl) propane-1,1-diphosphonic, e.g. EB 1053 (Leo); acid 1- (N-phenylaminothiocarbonyl) methane-1,1-diphosphonic, e.g. FR 78844 (Fujisawa); acid 5-benzoyl-3,4-dihydro-2H-pyrazol-3,3-diphosphonic tetraethyl ester, e.g. U-81581 (Upjohn); acid 1-hydroxy-2- (imidazo [1,2-a] pyridin-3-yl) ethane-1,1-diphosphonic, e.g. YM 529; and 1,1-dichloromethane- 1,1-diphosphonic (clodronic acid), e.g. clodronate

The uses of the present invention represent a improvement against existing therapy for malignant diseases in which bisphosphonates are used to prevent or inhibit development of bone metastases or excessive bone resorption, and also for the therapy of inflammatory diseases such as arthritis rheumatoid and osteoarthritis. It has been found that the use of bisphosphonates to embolize newly formed blood vessels leads to tumor suppression, for example, solid tumors, and of metastasts, for example, of bone metastasts, and even reduction in tumor size, for example, solid tumors, and metastasts, for example, bone metastasts, after appropriate periods of treatment. It has been observed, using angiography, that the vessels Newly formed blood disappear after treatment with bisphosphonate, but normal blood vessels remain intact In addition, it has been observed that blood vessels embolized are not restored after stopping treatment with bisphosphonate It has also been observed that patients of bone metastasis, rheumatoid arthritis and osteoarthritis, experience a decrease in pain following the bisphosphonate treatment.

Although the mode of action of bisphosphonates as causative agents of embolism of fresh blood vessels formed is not known, it seems that the blood vessels just formed (capillaries) are blocked, partially or completely destroyed, or angiogenesis is reversed in some way, leading to a partial or total disappearance of blood vessels newly formed (capillaries), for example, when the tumor or site of the disorder, that is, the site of inflammation, is observed using angiography. For the purpose of this description the terms "embolic treatment of angiogenesis" or "effect embolic "refer to these observed phenomena.

Accordingly, in an additional aspect, the invention provides the use of a bisphosphonate in the preparation of a medication as defined above for treatment embolic of angiogenesis.

In addition, as described in what follows in the Examples, it has been found that certain bisphosphonates inhibit angiogenesis induced by growth factor in a model animal and also inhibit endothelial cell proliferation in a tissue culture model. It seems that such inhibition of angiogenesis does not depend on the decay of activated macrophages but rather that bisphosphonate seems to act at the level of endothelial cell activation and / or cell proliferation endothelial

Thus, advantageously bisphosphonates can also be used for prophylactic or preventive treatment of diseases and medical conditions that involve angiogenesis, inhibiting the presence or development of angiogenesis, including diseases and medical conditions as identified previously.

Accordingly, in an even additional aspect The invention provides:

- use of a bisphosphonate in the preparation of u medication as defined above, for treatment prophylactic or preventive angiogenesis.

In particular, the invention provides a use as just defined, which does not depend on or involves the Decay of activated macrophages.

Thus, in the present description the terms "treatment" or "treatment" refers to both treatment prophylactic as preventive as well as curative treatment or disease modifier, including patient treatment at risk of contracting the disease or suspected of having contracted the disease as well as patients who are sick or have been diagnosed to suffer from a disease or condition medical

The bisphosphonates used in the present invention They are as described above.

Pharmaceutically acceptable salts of bisphosphonates for use in the invention are preferably salts with bases, conveniently metal salts derived from the groups Ia, Ib, IIa and IIb of the Periodic Table of the Elements, including alkali metal salts, e.g. potassium salts and especially sodium salts, or alkaline earth metal salts, preferably calcium or magnesium salts, and also salts of Ammonia with ammonia or organic amines.

Pharmaceutically acceptable salts especially preferred are those where one, two, three or four, in particular one or two of the acidic hydrogens of the acid bisphosphonate are replaced by a pharmaceutically cation acceptable, in particular sodium, potassium or ammonium, first sodium instance.

A very preferred group of pharmaceutical salts acceptable is characterized by having an acidic hydrogen and a pharmaceutically acceptable cation, especially sodium, in each one of the phosphonic acid groups.

All bisphosphonate acid derivatives mentioned above are well known in the literature. This includes its manufacture (see, for example, EP-A-513760, pp. 13-48). For example, the acid 3-amino-1-hydroxypropane-1,1-diphosphonic be prepared as described in US Patent 3,962,432 as well as the disodium salt in US patents 4,639,338 and 4,711,880, and the acid 1-hydroxy-2- (imidazol-1-yl) ethane-1,1-diphosphonic it is prepared as described, for example, in US Pat. 4,939,130.

A particular embodiment of the invention is represented by the use of a bisphosphonic acid derivative selected from acid 3-amino-1-hydroxypropane-1,1-diphosphonic, acid 3- (N, N-di-methylamino) -1-hydroxypropane-1,1-diphosphonic; acid 4-amino-1-hydroxybutane-1,1-diphosphonic; acid 6-amino-1-hydroxyhexane-1,1-diphosphonic, acid 3- (N-methyl-N-n-pentylamino) -1-hydroxypropane-1,1-diphosphonic; acid 1-hydroxy-2- (imidazol-1-yl) ethane-1,1-diphosphonic; acid 1-hydroxy-2- (3-pyridyl) ethane-1,1-diphosphonic, and N-methyl pyridinium salts thereof; acid 1- (4-chlorophenylthio) methane-1,1-diphosphonic; acid 3- [N- (2-phenylthioethyl) -N-methylamino] -1-hydroxypropane- 1,1-diphosphonic; acid 1-hydroxy-3- (pyrrolidin-1-yl) propane-1,1-diphosphonic; 1- (N-phenylaminothiocarbonyl) acid methane-1,1-diphosphonic; acid 5-benzoyl-3,4-dihydro-2H-pyrazol-3,3-diphosphonic tetraethyl ester; 1-hydroxy acid 2- (imidazo [1,2-a] pyridin-3-yl) ethane-1,1-diphosphonic; or to a pharmaceutically acceptable salt thereof, and any hydrate thereof.

A preferred embodiment of the invention is represented by the use of a bisphosphonic acid derivative selected from acid 3-amino-1-hydroxypropane-1,1-diphosphonic; acid 3- (N, N-di-methylamino) -1-hydroxypropane-1,1-diphosphonic; acid 4-amino-1-hydroxybutane-1,1-diphosphonic acid; 6-amino-1-hydroxyhexane-1,1-diphosphonic; acid 3- (N-methyl-N-n-pentylamino) -1-hydroxypropane-1,1-diphosphonic; acid 1-hydroxy-2- (imidazol-1-yl) ethane-1,1-diphosphonic; acid 1-hydroxy-2- (3-pyridyl) ethane-1,1-diphosphonic; acid 3- [N- (2-phenylthioethyl) -N-methylamino] - 1-hydroxypropane-1,1-diphosphonic; acid 1-hydroxy-3- (pyrrolidin-1-yl) -propane-1,1-diphosphonic; acid 1-hydroxy-2- (imidazo [1,2-a] pyridin-3-yl) -ethane-1,1-diphosphonic; or a pharmaceutically acceptable salt thereof, and any hydrate thereof.

A very preferred embodiment of the invention is represented by the use of a phosphonic acid derivative selected from pamidronic acid, alendronic acid, acid 3- (N-methyl-N-n-pentylamino) -1-hydroxypropane-1,1-diphosphonic; acid 1-hydroxy-2- (imidazol-1-yl) ethane-1,1-diphosphonic; risedronic acid and tiludronic acid; or a pharmaceutically salt acceptable thereof, and any hydrate thereof. A Especially preferred embodiment of the invention is represented. by the use of a bisphosphonic acid derivative, selected between acid 1-hydroxy-2- (imidazol-1-yl) ethane-1,1-diphosphonic and acid 3-amino-1-hydroxypropane-1,1-diphosphonic, or a pharmaceutically acceptable salt thereof, and any hydrate thereof

In addition, the invention relates to the use of acid 3-amino-1-hydroxypropane-1,1-diphosphonic or a pharmaceutically acceptable salt thereof or any hydrate thereof, for example, disodium pamidronate or pamidronate.

In addition, the invention relates to the use of acid 1-hydroxy-2- (imidazol-1-yl) ethane-1,1-diphosphonic or a pharmaceutically acceptable salt thereof or a hydrate of same, for example, zoledronic acid.

It has been found in an animal model, okay with the present invention, that zoledronic acid (acid 1-hydroxy-2- (imidazol-1-yl) ethane-1,1-diphosphonic)  preferentially inhibits angiogenesis induced by the factor of basic fibroblast growth (bFGF) compared to its inhibition of growth factor induced angiogenesis vascular endothelial, as described later in the Examples

Accordingly, in modalities particularly preferred the invention provides a use as defined above - in which the bisphosphonate is acidic zoledronic or a pharmaceutically acceptable salt thereof or a hydrate thereof and in which the angiogenesis comprises the bFGF-induced angiogenesis, or - in which the bisphosphonate is zoledronic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof and bisphosphonate is used in combination with a VEGF inhibitor

Bisphosphonates (Hereafter called Agents of the Invention) can be used in the form of a isomer or a mixture of isomers when appropriate, typically as optical isomers such as enatiomers or diastereoisomers or geometric isomers, typically isomers cis-trans. The optical isomers are obtained in the pure antipode form and / or as racemates.

Agents of the Invention can also be used in the form of your hydrates or include other solvents used for your recrystallization

Agents of the Invention (bisphosphonates) they are preferably used in the form of pharmaceutical compositions that contain a therapeutically effective amount of ingredient optionally active together with or in a mixture with vehicles inorganic or organic, solid or liquid, pharmaceutically acceptable appropriate for administration.

The pharmaceutical compositions can be, by example, compositions for enteral administration, such as oral, rectal, aerosol or nasal inhalation, compositions for parenteral administration, such as intravenous or subcutaneous, or compositions for transdermal administration (eg passive or iontophoretic).

Preferably, the pharmaceutical compositions are adapted for oral or parenteral administration (especially intravenous, intraarterial or transdermal). The intraarterial and oral administration, first, and primarily the intra-arterial administration, are considered of particular importance. Preferably, the active ingredient bisphosphonate, is in parenteral form, more preferably in form intraarterial

The particular mode of administration and dose can be selected by the attending physician, taking into tell the particular details of the patient, especially age, weight, lifestyle, hormonal status (for example, post-menopausal) and bone mineral density as appropriate. More preferably, however, the bisphosphonate is administered intraarterially in an artery leading to the site of the newly formed blood vessels.

Thus, in particularly preferred embodiments the invention provides:

Use of a bisphosphonate in the preparation of a medication as defined above for treatment intraarterial embolic angiogenesis.

The dose of the Agents of the Invention may depend on various factors, such as effectiveness and duration of the action of the active ingredient, mode of administration, species of warm blood, and / or sex, age, weight and individual condition of the warm blooded animal.

Normally, the dose is such that in one dose single active ingredient bisphosphonate 0.002 - 3.40 mg / kg, especially 0.01 - 2.40 mg / kg, it is administered to an animal of warm blood that weighs approximately 75 kg. If desired, this dose can also be taken in several doses, optionally same.

"mg / kg" means the mg of drug per kg of body weight of the mammal - including man - that is going to be treaty.

The aforementioned dose - well administered as a single dose (which is preferred) or in several partial doses- it can be repeated, for example, once a day, once a week, once every month, once every three months, once every six  months or once a year. In other words, the compositions Pharmaceuticals can be administered in regimens that vary from continuous daily therapy to clinical therapy intermittent.

Preferably, bisphosphonates are administered in doses of the same order of magnitude of those used in the treatment of diseases classically treated with derivatives of bisphosphonic acid, such as Pager's disease, Tumor-induced hypercalcemia or osteoporosis. In others words, bisphosphonic acid derivatives are administered in doses that would probably be therapeutically effective in Paget's disease treatment, induced hypercalcemia by tumors or osteoporosis, that is, preferably they are administered in doses that would probably be effective in inhibiting bone resorption

Formulations in unit dosage form simple preferably contain about 1% to approximately 90%, and formulations that do not fit single dose unit preferably contain from about 0.1% to about 20% of the active ingredient. Single dose unit forms such as capsules, tablets or dragees contain, for example, about 1 mg at approximately 500 mg of the active ingredient.

Pharmaceutical preparations for enteral and parenteral administration are, for example, those in unit dosage form, such as dragees, tablets or capsules and also ampoules. They are prepared in a manner known per se , for example, by means of conventional processes of mixing, granulation, confection, dissolution or lyophilization. For example, pharmaceutical preparations for oral administration can be obtained by combining the active ingredient with solid carriers, where appropriate by granulating a resulting mixture, and processing the mixture or granulate, if desired or necessary after the addition of appropriate adjuncts, in tablets or dragee cores.

Acceptable vehicles are especially agents of filler, such as sugars, for example, lactose, sucrose, mannitol or sorbitol, cellulose preparations and / or phosphates of calcium, for example tricalcium phosphate or hydrogen phosphate of calcium, and also binders, such as starch pastes, using, for example, corn, wheat, rice or potato starch, jelly, tragacanth, methylcellulose and / or polyvinylpyrrolidone, agar or acid alginic or a salt thereof, such as sodium alginate.

Attachments are especially regulatory agents of the flow and lubricants, for example, silicic acid, talc, acid stearic acid or salts thereof, such as magnesium stearate or calcium, and / or polyethylene glycol. Dragee cores are provided of appropriate coatings that can be resistant to juices gastric, being used in such cases, among others, solutions sugar concentrates that optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or dioxide titanium, or solutions of lacquered in solvents or mixtures of suitable organic solvents or, to produce coatings that are resistant to gastric juices, preparation solutions appropriate cellulose, such as acetyl cellulose phthalate or hydroxypropyl methylcellulose phthalate. Substances can be added dyes or pigments to tablets or coatings of dragees, for example, for identification purposes or to indicate Different doses of active ingredient.

Other administrable pharmaceutical preparations orally they are dry filled capsules made of gelatin, and also soft, sealed capsules, made of gelatin and a plasticizer, such as glycerol or sorbitol. The filled capsules dry can contain the active ingredient in the form of a granulated, for example in admixture with fillers, such as lactose, binders such as starches and / or glidants such as magnesium stearate, and, when appropriate, stabilizers. In soft capsules the active ingredient is preferably dissolved or suspended in appropriate liquids, such as oils fatty, paraffin oil or liquid polyethylene glycols, being It is also possible to add stabilizers. Formulations parenteral are especially injectable fluids that are effective in various ways, such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, subcutaneous or preferably intraarterial. Such fluids are preferably isotonic aqueous solutions or suspensions that they can be prepared before use, for example, from lyophilized preparations that contain the active ingredient well alone or together with a pharmaceutically acceptable vehicle. The Pharmaceutical preparations may be sterilized and / or contain Attachments, for example, preservatives, stabilizers, agents humectants and / or emulsifiers, solubilizers, salts to regulate Osmotic pressure and / or regulators.

Formulations suitable for application Transdermal include an effective amount with vehicle. Vehicles advantageous include pharmaceutically absorbable solvents acceptable to assist in the passage through the skin of the wearer. Normally, transdermal devices are in the form of a bandage comprising a back member, a reservoir that contains the compound optionally with conveyors, optionally a controlling barrier to release the active ingredient towards the carrier skin at a controlled and predetermined rat during a prolonged period of time, and means to secure the device To the skin.

The following Examples illustrate the invention that just described and, in relation to Example 5, refer the accompanying figures like this:

Figure 1 showing angiograms of the left bronchial arteries of a patient with breast cancer before (a) and after (b) pamidronate treatment disodium

Figure 2 showing angiograms of the right bronchial arteries of the same patient with cancer breast before (a) and after (b) pamidronate treatment disodium

Figure 3 showing knee angiograms right of the knee joint of a patient with osteoarthritis before (a) and after (b) treatment with disodium pamidronate.

In the following Examples the term "active ingredient" should be understood as one of the derivatives of bisphosphonic acid mentioned above as useful according to the present invention.

Examples Example 1

Capsules containing ingredient granules active coated, for example, disodium pamidronate pentahydrate As active ingredient.

one

A mixture of pamidronate disodium with Avicel® PH 105 it is wetted with water, agglomerated, extruded and formed into spheres. The dried granules are then coated successively in the bed fluidized with an internal coating, consisting of cellulose HP-M 603, polyethylene glycol (PEG) 8000 and talc, and the aqueous coating resistant to gastric juices, consistent of Eudragit® L 30 D, triethyl citrate and Antifoam® AF. The coated granules are powdered with talcum powder and filled in capsules (capsule size 0) by means of a filling machine of commercial capsules, for example, Höfliger and Karg.

Example 2

Monolithic adhesive transdermal system, which contains as an active ingredient, for example, acid 1-hydroxy-2- (imidazol-1-yl) -ethane-1,1-diphosphonic:

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Composition Polyisobutylene (GDP) 300 5.0 g (Oppanol B1, BASF) GDP 35000 3.0 g (Oppanol B10, BASF) GDP 1200000 9.0 g (Oppnol B 100, BASF) hydrogenated hydrocarbon resin 43.0 g (Escorez 5320, Exxon) 1-dodecylazacycloheptan-2-one 20.0 g (Azona, Nelson Res., Irving, CA) active ingredient 20.0 g Total 100.0 g

Preparation

The previous components dissolve together in 150 g of a fraction of special boiling point of oil 100-125 rolling them on a rolling bed. The solution is applied to a polyester film (Hostaphan, Kalle) by means of a spreading device using a medical foil 300 mm, producing a coating of approximately 75 g / m2. After drying (15 minutes at 60 ° C), a film of silicone treated polyester (thickness 75 mm, Laufenberg) is applied  As a removable film. The finished systems are cut in desired sizes from 5 to 30 cm 2 using a tool cut. The complete systems are individually sealed in bags of aluminized paper.

Example 3

A vial containing 1.0 mg of acid 1-hydroxy-2- (imidazol-1-yl) ethane-1,1- dry, lyophilized diphosphonic (sodium salts thereof). After to dilute with 1 ml of water, a solution is obtained (concentration 1 mg / ml) for infusion i.v.

Composition Active ingredient (free diphosphonic acid) 1.0 mg Mannitol 46.0 mg Citrate trisodium x 2H 2 O ca. 3.0 mg Water 1 ml Water for injection 1 ml

In 1 ml of water, the active ingredient is titled with trisodium citrate xH2O at pH 6.0. The mannitol is added immediately and the solution is lyophilized and the lyophilisate is deposited in a vial

Example 4

A blister that contains active ingredient, for example disodium pamidronate pentahydrate dissolved in water. The solution (concentration 3 mg / ml) is for infusion i.v. after dilute.

Composition Active ingredient ( ? 5.0 mg of active anhydrous ingredient) 19.73 mg Mannitol 250 mg Water for injection 5 ml

Example 5 Patient Treatment

A certain number of patients suffering from associated cancer and metastasis a patient suffering from Osteoarthritis are treated intraarterially with infusions of bisphosphonate through arteries leading to the site of the cancer, metastasis or osteoarthritis. The sites with Cancer, metastasis or osteoarthritis are examined using techniques angiographic both before and after infusion with bisphosphonate In all cases a marked effect is observed embolic on newly formed capillary vessels and other vessels blood in the region. The treatment regimens are described in more detail below:

i)

ID of patient 1676 Sex Female Age 68th 6 m Diagnosis Breast Cancer, Metastasis multiple in lung Pedicles therapeutic Bilateral bronchial arteries

A total of 75 mg of infusion is applied disodium pamidronate (Aredia®) in bronchial arteries bilateral to destroy their tumor eruptions. Figures 1 and 2 angiogram samples of the left lung areas (Figure 1) and right (Figure 2) before 8th) and after (b) the treatment.

ii)

ID of patient 2022 Sex Female Age 57th 1 m Diagnosis Breast Cancer, Metastasis bone, multiple pulmonary metastases Pedicles therapeutic 1. bilateral intercostal aretrias greater than 12th intercostal artery 2. bilateral bronchial arteries 3. bilateral internal thoracic artery Four. lateral thoracic artery vr.

A total of 75 mg pamidronate is used disodium (Aredia®), 100 mg of etoposide, 10 mg of BLM and 500 mg of impras-liprodol, to obliterate rashes tumors located along these pedicles (for bone metastasis is used only pamidronate).

iii)

ID of patient 1441 Sex Female Age 63rd 11 m Diagnosis Breast Cancer, Metastasis multiple bone, liver metastasis multiple Therapeutic pedicles 1. arteries bilateral shutters 2. arteries bilateral lumbar L 4/5 to L1 3. right hepatic subergemenales hepatic arteries 6, 7, 8.

A total of 45 mg pamidronate is used disodium (Aredia®), 10 mg of ADM (for liver) and 500 mg of as-liprodol impr emulsion, to destroy the tumor rashes located along these pedicles (For bone metastases, 45 mg of pamidronate is used only and 10 mg of ADM is additionally used for liver metastases with 500mg of impr as-liprodol.

iv)

ID of patient 1840 Sex Male Age 43rd 11 m Diagnosis Tongue cancer, vibo collateral, diaphragm metastasis Pedicles therapeutic 1. intercostal arteries 7, 8, 8, 10th. 2. Frenia it. Ing. 3. superior intercostal artery Item. 4. subcapsular artery it.

A total of 60 mg of infusion is applied disodium pamidronate (Aredia®) in the above arteries for destroy their tumor eruptions located along these pedicles

v)

ID of patient 1835 Sex Female Age 34th 8 m Diagnosis Breast Cancer, Metastasis multiple lung, liver metastases multiple Pedicles therapeutic  \ begin {minipage} [t] {110mm} It. Íleo lumbar, sacral ileus, lateral sacral arteries, lumbar ileus rt, bilateral deep iliac arteries L4, t L3, bilateral L2, thoracic artery 1, thoracic rt intercostal arteries, bronchial, hepatic artery rt. \ end {minipage}

A total of 90 mg of disodium pamidronate (Aredia®), 100 mg of etoposide, 10 mg of ADM, 6.0 CE of OK-432, and 500mg of emulsion impr as-liprodol are used to destroy rashes tumors located along these pedicles.

saw)

ID of patient 2013 Sex Female Age 73a 7 m Diagnosis  \ begin {minipage} [t] {110mm} Osteoarthritis of the right knee joint (interpreted as ischemic angiogenic bone disorder) \ end {minipage} Pedicles therapeutic 1. descending genicular artery right 2. right sular artery

A total of 30 mg pamidronate is used disodium (Aredia®) to destroy angiogenic eruptions located along these pedicles. The angiograms of the area of the right knee joint are shown in Figure 3 before 8a) and (b) after treatment.

vii)

ID of patient 2026 Sex Female Age 49th Diagnosis Cancer sinus, thoracic spine, bone metastasis Pedicles therapeutic 1. superior intercostal arteries bilateral 2. Frenia it. Ing. 3. bilateral lumbar arteries 1-4. 4. sacral artery half.

A total of 30 mg of infusion was applied disodium pamidronate (Aredia®) in tumor rashes to destroy them

viii)

ID of patient 1985 Sex Female Age 49th 8 m Diagnosis Breast Cancer, Metastasis multiple bone after BCT Pedicles therapeutic  \ begin {minipage} [t] {110mm} 1. arteries bilateral upper intercostals to the arteries intercostals 12 ^ a \ end {minipage} 2. arteries bilateral lumbar (L1 to L4). 3. artery middle sacral 4. sacral artery half.

A total of 90 mg pamidronate was used disodium (Aredia®), to destroy tumor eruptions located along these pedicles.

ix)

ID of patient 1063 Sex Male Age 80th 10 m Diagnosis Lung cancer, cancer squamous cell Therapeutic pedicles one. upper right intercostal arteries 2. common trunk of bilateral bronchial arteries

A total of 45 mg of infusion is applied disodium pamidronate only, in the tumor to destroy the tumor eruptions of the feeder pedicles.

Example 6 Effect of zoledronic acid on angiogenesis induced in mice by subcutaneous implants impregnated with the factor of increase Methods Animals

Female mice (Tiflbm: MAG) with weight were used from 17 to 20 g. They were identified by ear tags and kept in groups (6 animals per cage) under conditions normal and were observed daily. 6 mice were used per group of treatment in each experiment. All experiments are They performed at least twice.

Camera preparation

Porous tissue chambers were made of perfluoro-alkoxy-Teflon (Teflon®-PFA, 21 mm x 8 mm diameter, 550 ml volume) and perforated with 80 holes of 0.8 mm spaced regularly. Both ends They were sealed with removable covers of the same material. Be installed four to six cameras in a silicone tube (12 mm diameter). Then both ends of the tube were sealed with silicone rubber and 24 hours later the tube containing the Chambers were autoclaved (121 ° C, 15 min).

Growth Factors

The chambers were filled (total volume 0.5 ml) under sterile conditions, while still inside the silicone tube, with 0.8% w / v agar (BBL® Nr. 11849, Becton Dickinson, Meilan, France) with a content of 20 U / ml of heparin (Novo Nordisk A / S, 2880 Bagsvaerd, Denmark) and with or without human growth factor (human VEGF 2 mg / ml or human bFGF 0.3 mg / ml). The agar solution was maintained at 42 ° C before the
fill.

Camera implantation

Anesthesia was induced in the mice by 3% isoflurane inhalation (Forene®, Abbott AG, Cham, Switzerland) in oxygen. The chamber was implanted under aseptic conditions to through a small incision in the animal's back. The incision on the skin it was closed by staples (Autoclip 9 mm, Clay Adams).

Chamber Removal

Five days after implantation, the animals were anesthetized (3% isoflurane) and slaughtered using an overdose of pentobarbitone (210 mg / kg i.p., Vetanarcol, Veterinaria AG, Zürich, Switzerland). The cameras were extracted from the animal, the vascularized fibrous tissue that had formed around each implant was carefully removed, it weighed and the total amount of blood was quantified by colorimetric determination of hemoglobin concentration.

Quantification of the angiogenic response

After the addition of 2 ml of distilled water, tissue samples were homogenized for 1 min at 24,000 rpm (Ultra Turrax T25). The samples were then centrifuged for 1 h at 7000 rpm. The supernatant was filtered through a 0.45 mm GHP syringe filter (Acrodisc® GF, Gelman Sciences, Ann Arbor, Michigan, USA) to avoid contamination with fat. The Amount of hemoglobin present in the filtrate was determined by 540 nm spectrophotometric analysis using reagent kit Drabkin (Sigma hemoglobin # 525, Sigma Chemical Co. Ltd., Poole, Dorset, England). An aliquot of the filtrate (100 ml) was added to 1 ml of Drabkin's solution and the mixture was incubated for 15 min at room temperature. Absorbance at 540 nm (Uvikon 810 P) It is proportional to the hemoglobin concentration. The measures of hemoglobin were then converted to volume measurements blood (ml) using a previously obtained calibration curve with whole blood samples of different volumes obtained from A donor mouse

Drug treatment

The administration of zoledronic acid began one day before the implantation of the cameras, and the cameras were removed 24 hours after the last dose, 5 days after implantation. The animals were caged by groups for the duration of the experiment. Zoledronic Acid It was supplied in doses of 1, 10 and 100 mg / kg / day s.c. (volume of dose 25 ml / kg) with injection applied at alternate sites. He compound was dissolved first in distilled water and then diluted with distilled water containing mannitol (final concentration: 5% of mannitol) Control animals received only the vehicle. Every experiment was carried out at least twice and then it consolidated for the final evaluation.

Calculations and Statistics

The percentage of response inhibition angiogenic (increased weight of tissue or whole blood) is calculated on individual animals as follows:

(A-B) / (C-D) x 100

A = weight (or volume of blood) of the tissue of a mouse treated with the drug with a camera containing factor of increase.

B = average weight (or volume of blood) of tissue from the group of mice treated with the drug with cameras It didn't contain the growth factor.

C = average weight (or volume of blood) of tissue from the group of mice treated with vehicle with cameras that They contained the growth factor.

D = average background weight (or blood volume) of the tissue of the group mice treated with the vehicle with cameras that did not contain the growth factor.

Statistical comparisons between groups (animals treated with the untreated compound) are carried out on the absolute values of tissue weight and blood volume using the sum of ranks test of Mann-Whitney (SigmaStat 2.0 program, Jandel Scientific, Germany). The significant level was set at p <0.05.

Results

Zoledronic acid, depending on the dose, inhibited the angiogenic response induced by bFGF, according to measurements of weight and blood content, with IC50 values Approximately 2.5 and 3.1 mg / kg, respectively. In the highest dose tested (100 mg / kg / day), there was a significant inhibition of increase in blood content induced by VEGF, but not in the weight.

These studies show that the acid zoledronic inhibits bFGF but not angiogenesis induced by VEGF in a dose range that shows therapeutic effects in the bone reconversion in various models of animal diseases. At higher doses, a partial inhibition of the VEGF response. Thus, zoledronic acid seems to have some selectivity for inhibition of the response mediated by the bFGF.

Example 7 Effects of Zoledronate on cell proliferation and in vitro migration Methods Compounds and test solutions

The studies described in this report were carried out with the hydrated disodium salt of zoledronate. Be prepared stock solutions of zoledronate (10 mM) and EDTA (5 mM) in PBS Then the stock solutions were diluted in the optimal medium for Each cell line.

Cells and cell culture conditions

Human umbilical vein endothelial cells were obtained from Promo Cell (BioConcept AG, Allschwil, Switzerland). The human tumor cell line, A431 (epithelial carcinoma) used in this study was obtained from the Type Culture Collection (ATCC, Rockville, MD, USA). All cell lines were cultured in vitro according to the recommendations of the provider.

Endothelial Cell Proliferation Assay

As a test of zoledronate's ability to inhibit a functional response to VEGF, a test of endothelial cell proliferation, based on the incorporation of BrdUAs (Biotrak Cell Proliferation Elisa System V.2, Amersham, England). To test the specificity of the response, also the effects of zoledronate and EDTA were evaluated (as a control for the chelating effects of zoledronate ions) on bFGF and HUVEC induced cell proliferation. Subconfluent HUVEC it was seeded at a density of 5 x 10 3 cells per well in 96-well plates with 1.5% gelatin and then incubated at 37 ° C

Y

5% CO2 in culture medium (Endothelial medium Cell Growth, PromoCell Nr. C-22110, BioConcept AG, Allschwil, Switzerland).

After 24 hours, the growth medium was replaced by a basal medium (Endothelial Cell Basal medium, PromoCellNr. C-22210, BioConcept AG, Allschwil, Switzerland) containing VEGF165 (10 ng / ml), human bFGF (0.5 ng / ml) or 5% FCS, in the presence or absence of the test compound. How control, wells without growth factor were also included. After 24 hours of incubation, the labeling solution was added of BrdU and the cells were incubated for 24 more hours before their antibody binding, blocking and addition anti-BrdU labeled with peroxidase. Antibody bound was detected using a substrate of 3,3,5,5'-tetramethylbenzidine, which forms a colored reaction product that is quantified spectrophotometrically at 450 nm.

Tumor Cell Proliferation Assay

To further test the proliferation of zoledronate and EDTA, its effects on the proliferation of a human tumor cell line (A431, carcinoma epithelial). The cells were seeded at 1.5 x 10 3 cells / well in 96-well plates and incubated overnight. The test compound was then added in serial dilutions and then the plates were incubated for 3 days, after which cells were fixed with 3.3% v / v glutaraldehyde, washed with water and stained with 0.05% w / v methylene blue. After washing, the dye was eluted with 3% v / v HCl and was measured the optical density at 665 nm with a Dynatech spectrophotometer 7000. The percentage reduction of cell cell growth exposed to the compound, compared to controls, was determined by a computerized system using the formula (OD test - OD start) / (OD control - OD start) x 100.

The IC50 was defined as the concentration of drug that leads to a 50% reduction in cell number per well compared to control crops (100%) at the end of incubation period.

Endothelial Cell Migration Assay

As proof of zoledronate's ability to inhibit a functional response to VEGF, a test of Endothelial cell migration. The plates were covered (24 wells) with 1.5% gelatin and circular fences were installed as a barrier to prevent cells from growing in the center from the well. HUVEC subconfludents were seeded in the external area with a density of 1 x 10 5 cells per well and incubated then at 37 ° C and 5% CO2 in the middle of (Endothelial Cell Growth medium, PromoCell

Nr. C-22110, BioConcept AG, Allschwil, Switzerland). After 24 hours, the fences were removed and growth medium was replaced by baseline (medium Endothelial Cell Basal, PromoCell Nr. C-22210, BioConcept AG, Allschwil, Switzerland) containing human VEGF165 (10 ng / ml) or 5% FCS, in the presence or absence of the test compound. To inhibit cell proliferation, 50 mg / ml of fluorouracil (Roche, Basel, Switzerland). As a control, I also know They included wells without growth factor. After 60-70 hours of incubation, the cells were fixed and stained with Diff-Quik (Dade Behring AG., Nr. 130832, Düdingen, Switzerland). 24 well plates were placed under a binocular microscope and the number of cells was counted migrated, using the KS-400 program (Carl Zeiss Jena, Jena, Germany) and a specially designed macro (migration.mcr).

Statistics

The data were analyzed by analysis of one-way variance followed by Dunnett's test to establish the significance of the differences between the groups treated with zoledronate or EDTA and control. The test of Bonferroni was used to establish the significance of the differences between each pair of zoledronate-treated groups and EDTA in a specific concentration. All the analyzes were carried out with the Instat® program (GraphPad Inc., San Diego, USA).

Results

Zoledronate in dose dependence, inhibited serum-induced HUVEC proliferation, with a value IC50 of 4.1 ± 0.6 mM. It also had a similar effect on the VEGF-induced proliferation (IC50: 6.9 ± 0.4 mM) and by bFGF (IC50: 4.2 ± 0.4 mM). The inhibitory effect of zoledronate on proliferation was significantly different from the control in the highest concentrations. For all stimuli, the Changes in cell morphology were observed with the Higher concentrations of zoledronate tested (30 mM).

Since bisphosphonates chelate the divalent cations, EDTA was used as a control in experiments parallel EDTA at 30 mM inhibited the proliferation of HUVEC induced by FCS, VEGF and bFGF by 23.7%, 55.6% and 49.5%, respectively. For all stimuli, the effect of EDTA was less than that of zoledronate, it did not reach statistical significance for serum-induced proliferation within the range of concentration tested, while it did with stimulation with VEGF and bFGF in EDTA concentrations of 3 nM and higher. For serum or bFGF stimulation, the inhibitory effect of zoledronate was significantly higher than that of EDTA in concentrations of 3 mM and higher, but with VEGF stimulation only 30 mM values were significantly different.

The proliferation of the tumor cell line human was inhibited by zoledronate with an IC50 of 1.35 mM.

EDTA had no significant effect on the proliferation of A431 in the dose range tested, although it detected a slight inhibition (8.8%) at 30 mM.

Zoledronate in concentrations up to 10 mM stimulated the migration of HUVEC in a basal environment as well as in a medium containing serum or VEGF. At 30 mM the migration was completely inhibited and a change in morphology was observed mobile. EDTA had no consistent effects on migration. These studies show that zoledronate inhibits the serum-induced human endothelial cell proliferation, VEGF and bFGF.

Claims (7)

1. Use of a bisphosphonate in the preparation of a medication for the treatment of myocardial ischemia, osteoarthritis, psoriasis, hemangioblastoma, hemangioma or pain, in where bisphosphonate acts as an inhibitory agent or reversal of angiogenesis and is selected from among following compounds or a pharmaceutically acceptable salt of the same, or any hydrate thereof: acid 3-amino-1-hydroxypropane-1,1-diphosphonic  (pamidronic acid), e.g. pamidronate (APD); acid 3- (N, N-dimethylamino) -1-hydroxypropane-1,1-diphosphonic, e.g. dimethyl-APD; acid 4-amino-1-hydroxybutane- 1,1-diphosphonic (alendronic acid), e.g. alendronate; acid 1-hydroxy-etidene bisphosphonic, e.g. etidronate; acid 1-hydroxy-3- (methylpentylamino) -propylidene bisphosphonic, ibandronic acid, e.g. ibandronate; acid 6-amino-1-hydroxyhexane-1,1-diphosphonic, e.g. amino-hexyl-BP; acid 3- (N-methyl-N-n-pentylamino) -1-hydroxypropane-1,1-diphosphonic, e.g. methyl-pentyl-APD (= BM 21.0955); acid 1-hydroxy-2- (imidazol-1-yl-ethane-1,1-diphosphonic, e.g. Zoledronic acid; acid 1-hydroxy-2- (3-pyridyl) ethane-1,1-diphosphonic (risedronic acid), e.g. risedronate, including salts N-methyl pyridinium thereof, for example, N-methyl pyridinium iodides such as NE-10244 or NE-10446; acid 1- (4-chlorophenylthio) methane-1,1-diphosphonic (tiludronic acid), e.g. tiludronate; acid 3- [N- (2-phenylthioethyl) -N-methylamino] -1-hydroxypropane-1,1-diphosphonic;  acid 1-hydroxy-3- (pyrrolidin-1-yl) propane-1,1-diphosphonic, e.g. EB 1053 (Leo); acid 1- (N- phenylaminothiocarbonyl) methane-1,1-diphosphonic, e.g., FR 78844 (Fujisawa); acid 5-benzoyl-3,4-dihydro-2H-pyrazol-3,3-diphosphonic tetraethyl ester, e.g. U-81581 (Upjohn); acid 1-hydroxy-2- (imidazo [1,2-a] pyridin-3-yl) ethane-1,1-diphosphonic, e.g. YM 529; Y 1,1-dichloromethane-1,1-diphosphonic acid (clodronic acid), e.g. clodronate 2. A use according to claim 1, in which bisphosphonate is used in the preparation of a medicine for the embolic treatment of angiogenesis. 3. A use according to claim 1, in which bisphosphonate is used in the preparation of a medicine for the prophylactic or preventive treatment of angiogenesis 4. A use according to claim 1, in which bisphosphonate is used in the preparation of a medicine for the treatment of angiogenesis in a patient suffering from myocardial ischemia or osteoarthritis. 5. A use according to claim 1, in which bisphosphonate is pamidronic acid or zoledronic acid, or a pharmaceutically acceptable salt thereof, or a hydrate of the same. 6. A use according to claim 1, in which bisphosphonate is used in the preparation of a medicine intraarterial embolic angiogenesis. 7. A use according to claim 6, in which bisphosphonate is pamidronic acid or zoledronic acid, or a pharmaceutically acceptable salt thereof, or a hydrate of the same.